All cells respond to stress such as heat shock, UV irradiation, and viral infection, and do so in part by altering gene expression. A critical control point for regulating gene expression in eukaryotic cells is at the level of mRNA transcription by RNA polymerase II. In a break with paradigm, the preliminary data discussed here identify a natural RNA, mouse B2 RNA, which can bind to and inhibit transcription by RNA polymerase II. The proposed studies will investigate the role of B2 RNA as a transcriptional repressor in response to cellular stresses including heat shock, DNA damage, and viral infection. Completion of these studies will provide insight into how transcription is regulated under conditions of stress, which is vitally important in understanding normal cell growth as well as aberrations associated with diseases and deleterious environmental conditions. The specific aims of the proposal are the following: 1) To study the function of B2 RNA as a negative regulator of the transcriptional response to heat shock, DNA damage, and viral infection in mouse cells. 2) To determine the molecular mechanism by which B2 RNA inhibits transcription by RNA polymerase II. 3) To map the regions of B2 RNA and RNA polymerase II that interact, and in doing so develop a small nucleic acid inhibitor of the transcriptional response to stress. 4) To characterize human RNAs that bind to RNA polymerase II and regulate transcription in response to heat shock, DNA damage, and viral infection. The specific aims utilize both in vitro experiments and cell-based assays. The in vitro experiments employ a purified RNA polymerase II transcription system as well as nuclear extracts prepared from stressed mouse cells. The cell-based experiments utilize techniques to decrease levels of regulatory RNAs in cells such as antisense, ribozyme, and RNAi, as well as techniques to monitor RNA polymerase II activity such as nuclear run-on assays and chromatin immunoprecipitations. The proposed experiments have the potential to show that natural small noncoding RNAs can regulate transcription by targeting RNA polymerase II, to experimentally demonstrate a function for the SINE encoded B2 RNA, and to propose a mechanism by which general mRNA transcription is repressed in response to cellular stress.